Biochemical, Biophysical, and Structural Mechanisms of HIV-1 Budding and Release Project Summary The budding and release of HIV from infected T-cells and macrophages is an essential step in the HIV replication cycle. HIV bud formation requires the virally encoded Gag polyprotein, and release requires the host-encoded ESCRT proteins. The ESCRTs sever narrow membrane necks, which occur physiologically in cytokinesis and in intralumenal membrane budding into endosomes. Our laboratory has purified and fluorescently labeled full-length HIV-1 Gag, all of the human ESCRT proteins, and capsids of the related Mason-Pfizer Monkey Virus (M-PMV). We reconstituted the ordered assembly of the human ESCRT proteins onto membrane-associated HIV-1 Gag clusters in vitro. Building on this unique set of reagents, the specific aims of this project are as follows: 1. To elucidate in biophysical detail the coupling between HIV-1 Gag assembly, lipid domain formation, RNA binding, and ESCRT recruitment. We will dissect the interplay between Gag assembly, lipid domain formation, RNA packaging, and ESCRT recruitment. Having analyzed the assembly of ESCRTs, we will go on to dissect their ATP-dependent disassembly by VPS4. 2. To reconstitute ESCRT-mediated release using a structurally tractable model virus. M-PMV buds from the plasma membrane via ESCRT-I, like HIV-1. M-PMV has the advantage that procapsids can be pre- assembled in solution, allowing us to decouple procapsid assembly from membrane binding and budding. We have developed M-PMV as a model system for in vitro reconstitution of ESCRT-dependent budding, and will use the system to obtain mechanistic insight at unprecedented spatial resolution. 3. To elucidate how TSG101, VPS28, and VPS37 and UMA protein isoforms assemble into the ESCRT-I complex. Of all the ESCRT components, the ESCRT-I complex has a central and essential role in the recruitment of the ESCRT machinery to HIV-1 budding sites. The presence of four VPS37 isoforms and up to five UMA proteins with different roles in endosomal and viral budding creates a complicated picture. The structure of a representative human ESCRT- I core complex will be determined and used as the starting point to understand subunit selectivity in ESCRT-I assembly and function in HIV budding.